For decades real-time Polymerase Chain Reaction (PCR) has been the gold standard for biothreat detection across the enterprise engaged in biodefense. Since its advent in 2005, Next Generation Sequencing (NGS) technologies have made significant strides in genomics, marching towards adoption into various realms of biology and medicine including diagnostics/detection. However, the ubiquitous use of NGS in diagnostics (Dx) and detection/identification (Di) applications, as does PCR, has not been fully realized and has only recently started. One of the premier technologies that accelerated this democratization is Oxford Nanopore Technology (ONT). ONT is portable, easy to use, requires minimal infrastructure to establish a sequencing capability, and has potential for use in austere environments.

In thinking about NGS-based biodetection, there are primarily two approaches: targeted and untargeted sequencing. The targeted (aka Amplicon Sequencing or Amp Seq) is relatively faster and less analytically intensive and easy to adopt in labs already doing PCR-based surveillance. Untargeted sequencing (Metagenome Sequencing or Meta Seq) has analytical challenges and is not ready for prime time yet, although that is the desired end goal. In addition, cost and time for metagenome sequencing can be prohibitive for routine surveillance. Hence, Amp Seq can be a bridge between PCR and Meta Seq.

Metagenome Sequencing is like finding a needle in a haystack

Unbiased, untargeted sequencing of samples for biothreat detection is the so-called metagenome sequencing also known by various other terms: threat agnostic sequencing; agnostic diagnostics; agent agnostic detection. Meta Seq is the one that changes the paradigm and can be used to identify unknown biothreats, i.e., newly emergent threats of natural origin or intentional/genetically modified threats that we have not seen before. However, it is like finding a needle in the haystack and comes with challenges. Meta Seq to identify a threat is not only akin to finding a needle in the haystack, but also which part of the needle we want to find. Needle being the threat organism and haystack being all else in a sample. Complex samples can contain millions of innocuous organisms and host, plant, and other nucleic acid materials. Finding the biothreat in this mix depends on a lot of factors. First, how much of threat organism is in the sample – the more of the threat organism present, the easier it would be to identify it at deeper taxonomic level. Secondly, at what taxonomic level (which part of the needle) you want to detect: PCR most often detects at genus/species level discrimination; we can also detect known genetic changes such as antimicrobial resistance (AMR) using a targeted PCR assay. If you want to drill further down into lower taxonomic levels below genus/species, it requires more initial biomass, more sequencing, more time and more complex post-sequencing analyses. Genetic modifications at single-nucleotide levels can have profound implications on phenotypes such as virulence and AMR.  However, sequencing to drill down to identify nucleotide-level variations is more complex and requires higher biomass of threat agent, some form of amplification of low-density organisms, or depletion of other genetic materials. Amplification, enrichment or depletion of background genetic material is going to be a targeted approach, i.e., you need a priori knowledge of what organisms to amplify or enrich, and, for depletion of the background, what are not the organisms of interest.

  

Nucleotide-level variant detection requires more time to sequence (not happening in minutes or few hours especially if the biomass is low). This longer time is needed to generate sequence data of better quality, with more breadth and depth of coverage and to process the data. It also entails having bioinformatic expertise on the back end and will likely be more expensive and may be prohibitive for routine daily use of biosurveillance. Nonetheless, Meta Seq may be useful in initial identification of a novel threat (discovery phase) and trigger early outbreak responses, such as developing a traditional real time PCR or an Amp Seq assay for routine specimen screening and implementing public health measures (e.g., SARS-CoV-2 pandemic response).

Read the full article in Global Biodefense magazine here.